Understanding normal human development depends on animal model systems for experimental insight into underlying biological mechanisms. The long-term goal of this project is to use cephalopod biology to understand human organogenesis, including brain development. Among animals, human beings are large organisms with disproportionately large brains. Cephalopods, which include squids, octopuses, and cuttlefish, are the largest non-mammalian marine organisms and have the largest brains among all invertebrates. To study cephalopod development at a mechanistic level requires the identification of a cephalopod with ready availability, easy husbandry, and an embryology permitting experimental manipulation. The investigators have identified such a preparation, the Californian mud-flat octopus, O. bimaculoides. To date, they have developed methods for ex ovo culturing and for whole mount in situ hybridization for embryonic stages from the blastoderm to hatching. The investigators have also isolated a large panel of cDNAs for key developmental control genes. To complete this model system, they need to develop reliable methods of gene manipulation in these cephalopod embryos. Specific Aim 1 is to generate cephalopod-specific expression vectors based on octopus EF1-a and ubiquitin C genes. Specific Aim 2 is to develop methods of plasmid delivery to octopus embryos, for which the investigators will test as methods electroporation and chemical transgenesis using cationic polymers. Development of gene manipulation resources and methods will permit hypothesis-driven research of the molecular and cellular mechanisms of cephalopod development. [unreadable] [unreadable] [unreadable]